African swine fever virus p72 recombinant protein, monoclonal antibody and test paper

Abstract

The invention provides an African swine fever virus p72 recombinant protein, a monoclonal antibody and test paper. An amino acid sequence of the African swine fever virus p72 recombinant protein is obtained by linking amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 through flexible amino acid fragments, and has strong immunogenicity and specificity. The invention also provides a nucleotide sequence for encoding the recombinant protein. The invention also provides the monoclonal antibody against the African swine fever virus p72 recombinant protein obtained by immunizing animals byusing the recombinant protein. The invention also provides hybridoma cells 7A7 and 3E5 with preservation numbers of CGMCC No. 18540 and CGMCC No. 18539, respectively, and the monoclonal antibody secreted by the hybridoma cells has high sensitivity and specificity to African swine fever virus p72. The invention also provides the latex microsphere test paper containing the recombinant protein antibody and the colloidal gold test paper containing the recombinant protein antibody, and the test paper has the advantages of small batch difference, high detection sensitivity and simple operation.

Description

technical field [0001] The invention belongs to the technical field of animal disease detection, and specifically relates to African swine fever virus p72 recombinant protein, nucleotide sequence, monoclonal antibody, hybridoma cells and test paper. Background technique [0002] African swine fever (ASF) is a hemorrhagic, highly fatal, viral infectious disease of pigs caused by African swine fever virus (ASFV). Pigs of all ages are susceptible. The incubation period of natural infection of ASF is long, ranging from 4 to 19 days. The average death time after infection is 2-10 days. The clinical manifestations include high fever, loss of appetite, skin and internal organ bleeding, etc. The clinical symptoms are similar to swine fever and swine danqing virus. After pigs or wild boars are infected with African swine fever virus, the incubation period is usually 3-15 days, and the mortality rate of virulent strains can reach 100%. For investigation and diagnosis in the early s...

AI Technical Summary

Problems solved by technology

In the binding reaction of antigen and antibody, the part where the antigen participates in the binding is called the epitope of the antigen. The epitope is the basis of the antigenicity of the protein. The epitope peptide commonly used in the current technology is still weak in immunogenicity and low in specificity. Therefore, there is an urgent need for a recombinant antigenic protein of African swine fever virus p72 with strong immunogenicity and good specificity, and the recombinant antigenic protein can be used to prepare monoclonal or polyclonal antibodies combined with it, and can be further utilized to obtain Antibodies for detection of African swine fever virus

[0004] In addition, the traditional methods for diagnosis and detection of African swine fever virus include molecular biology methods and enzyme-linked immunoimmunoassay kit methods. The molecular biology methods are represented by polymerase chain reaction (PCR), which confirms whether the animal is infected by detecting virus molecules. Infected, the operation is cumbersome, the reagents and equipment are expensive, and it is difficult to operate for pig farms; ELISA uses 96-well ELISA plate as the carrier, and uses the principle of antigen-antibody specific reaction to detect whether animals are infected with ASF virus, which is sensitive High, specificity, etc., the fly in the ointment is that the operation process is relatively cumbersome, the accuracy of sample addition is high, and experimental equipment such as microplate readers and incubators are needed to ensure the reaction environment, which is difficult to carry out in grassroots farms, especially on-site testing. ; None of the above methods can achieve the purpose of rapid detection

At present, the most widely used test strips are colloidal gold test strips, which also use the principle of antigen-antibody reaction to detect African swine fever virus antigens. The operation is simple and the reaction time is short, but the disadvantage is that the sensitivity is not high enough, and there may be false negatives or false positives. Especially for the rapid detection of ASFV, because the samples are mostly whole blood, tissue samples, etc., the sample components are complex, and it is impossible to carry out long-term pretreatment, which may cause problems such as interference and poor specificity of the test strips, and it is difficult to meet the requirements of rapid detection of ASFV

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